Alkylating and methylating agents are important groups of compounds for use in cancer chemotherapy. Chloroethylating agents, including but not limited to chloroethylnitrosoureas, form DNA adducts within the cell nucleus that promote alterations in DNA structure and/or function. These changes at the DNA level lead to cytotoxicity within the targeted cell.
Chloroethylnitrosoureas such as BCNU (N,N'-bis(2-cholorethyl)-N-nitrosourea) and CCNU (N-(2-cholorethyl)-3-cyclohexyl-N-nitrosourea) are lipid soluble compounds that have been shown to possess clinical utility against some neoplasms but with limited success in clinical trials. These chloroethylnitrosoureas promote DNA alkylation at the O.sup.6 position of guanine, leading to DNA interstrand crosslinking and altered fidelity of DNA replication and transcription. This induced interstrand crosslinking involves formation of a chloroethyl adduct at the guanine residue that undergoes an intramolecular rearrangement to produce an unstable intermediate that reacts with the cross strand cytosine residue. The result is a N.sup.1 -guanine, N.sup.3 -cytosine-ethanol crosslink.
This N.sup.1 -guanine, N.sup.3 -cytosine-ethanol crosslink can be prevented by the DNA repair protein, O.sup.6 -alkylguanine-DNA alkyltransferase (AGT). This repair protein is active in mammalian tumor cells and is responsible for protecting cells from the antitumor effects normally associated with chloroethylating agents such as BCNU and CCNU. AGT has a unique mechanism of action in that it brings about the transfer of alkyl groups present on the O.sup.6 position of guanine in DNA to a cysteine residue located within the AGT amino acid sequence (Lindahl, et al., 1988, Annu. Rev. Biochem. 57: 133-157; Pegg, 1990, Cancer Res. 50: 6119-6129). The resulting S-alkylcysteine-containing AGT protein is not converted back to cysteine. AGT acts only once and the number of O.sup.6 -alkylguanine residues that can be repaired is equal to the number of available AGT molecules. Therefore, tumor cells expressing high levels of AGT show resistance to alkylating and methylating chemotherapeutic drugs, which may limit clinical effectiveness of these agents. To this end, a reduction of functional AGT in mammalian tumor cells should correlate to increased sensitivity of these cells to the chemotherapeutic effects of chloroethylating agents that form DNA adducts at the O.sup.6 position of guanine.
U.S. Pat. No. 5,091,430, issued to Moschel, et al. on Feb. 24, 1992 discloses O.sup.6 -substituted guanine compounds which inhibit AGT. An exemplified compound which inhibits AGT is O.sup.6 -benzylguanine (BG). It has been shown that BG is a strong time and concentration dependent inactivator of human AGT (Dolan, et al., 1990, Proc. Natl. Acad. Sci. 87: 686-690). This mechanism has been confirmed by the identification of S-benzylcysteine in AGT and the formation of stoichiometric amounts of guanine following incubation with BG.
U.S. Pat. No. 5,352,669 issued to Moschel, et al. on Oct. 4, 1994 discloses O.sup.6 -substituted guanosine and 2' deoxyguanosine compounds which are shown to inhibit AGT.
U.S. Pat. No. 5,358,952 issued to Moschel, et al. on Oct. 22, 1994 disclose pharmaceutical combinations for chemotherapy comprising O.sup.6 -substituted guanine compounds in tandem with anti-neoplastic alkylating agents.
A major limitation in the use of alkylating and methylating agents in the treatment of neoplastic disease is the profound myelosuppression produced by these drugs. This problem peaks at about 4-6 weeks after treatment and thus prevents the repetition of cyclic therapy at preferred intervals. This myelosuppression is due to low concentrations of AGT in hematopoietic cells.
Crone and Pegg (1993, Cancer Res. 53: 4750-4753) disclose a mutant human AGT protein with a single amino acid change of proline to alanine at aa #140 (P140A). This mutant human AGT shows a decrease in sensitivity to BG in vitro. The authors do not address in vivo activity of this mutant in regard to BG or BG/BCNU combinations.
Crone, et al. (1994, Cancer Res. 54: 6221-6227) disclose an additional mutant human AGT protein with a single amino acid change of glycine to alanine at aa #156 (G156A). As with human AGT P140A, G156A shows a decrease in sensitivity to BG in vitro. Again, the authors do not address in vivo activity of this mutant in regard to BG or BG/BCNU combinations.
Gerson, et al. (1994, Mutation Res. 307:541-555) disclose transgenic mice expressing either the wild type human O.sup.6 -methylguanine-DNA methyltransferase (MGMT) cDNA or the bacterial ada gene (which expresses a prokaryotic version of AGT resistant to BG). The authors show a level of protection against addition of BCNU in transformed cell types shown to express the respective gene.
Another avenue of addressing alkylating agent toxicity is gene therapy. Moritz, et al. (1995, Cancer Res. 55:2608-2614) disclose retroviral mediated expression of MGMT in murine bone marrow cells. The authors used a strong promoter in attempts to overexpress MGMT in the cells in an effort to overcome the effect of added BCNU for in vitro and in vivo studies. No attempt was made nor suggested by the authors to inhibit MGMT at the tumor site and provide a resistant form of the protein in hematopoietic cells to reduce BCNU-induced myelosuppression. Allay, et al. (1995, Blood 85: 3342-3351) also show retroviral mediated expression of MGMT in murine bone marrow cells. The authors used the MPSV vector with the 5'LTR promoter fragment to increase expression of MGMT and also obtain in vitro reduction in BCNU-induced myelosuppression. Harris, et al. (1995, Proc. Amer. Assoc. Can. Res. 36: 419) disclose retroviral mediated expression of a bacterial ada gene and a measure of resistance to nitrosoureas. The authors noted increased resistance to BG and BCNU in vivo. Maze, et al. (1996, Proc. Natl. Acad. Sci. 93: 206-210) disclose in vitro and in vivo expression of wtMGMT in mouse bone marrow cells. These authors do not suggest inhibition of MGMT at the tumor site and providing a human resistant form of the protein in hematopoietic cells to reduce BCNU-induced myelosuppression. These studies lack an efficient gene therapy application at the clinical level since the routine high level expression of AGT in these cells is difficult to accomplish and even substantial increases in alkyltransferase may not be sufficient to render these cells less susceptible to killing than many of the tumors which are being treated since these tumors may have high levels of alkyltransferase. Although MGMT overexpression increases BCNU resistance in normal murine and human hematopoitic cells, the effect has been quite modest, leaving serious questions about any therapeutic utility for these applications. Therefore, a fundamental problem exists in relation to overexpression of human AGT in hematopoietic cells in an attempt to overcome sensitivity to chloroethylating compounds targeted to tumor cells.
Although AGT P140A and G156A show resistance to BG and no apparent defect in the ability to repair O.sup.6 -methylguanine in DNA in vitro, the rate of repair of methylated DNA is very rapid and is difficult to measure accurately under physiological conditions. Also, it is not known whether the ability to act on the larger 2-chloroethyl group is affected by these mutations. Furthermore, some point mutations in AGT have a pronounced destabilizing effect and may reduce the steady state level of the AGT protein. These factors could limit the ability of the mutant AGTs to protect cells from chloroethylation. Therefore, it is possible that some or all of these mutations do also affect the ability to repair O.sup.6 -(2-chloroethyl)guanine in cellular DNA and would therefore not produce resistance to sensitization by BG.
Therefore, despite attempts to overcome the extreme myelosuppression observed with the use of alkylating and methylating agents in chemotherapy regimes, a need exists for improved methods of using these anti-neoplastic agents to treat various neoplastic diseases.